One of the most important functions of endoplasmic reticulum (ER)-Golgi system is to facilitate post-translational modifications including protein folding. The correct folding of newly-translated proteins is essential for secreted extracellular proteins and membrane proteins to ensure that they can function correctly. Protein folding allows a polypeptide to fold into its characteristic and functional three-dimensional structure. Protein folding involves the formation of both non-covalent and covalent bonds. Generally in eukaryotic cells, protein folding is mediated in the rough ER by ER chaperone proteins.
Protein disulphide isomerase (PDI) is a chaperone protein which catalyzes the formation and isomerisation of disulphide bonds between two cysteine residues in polypeptides. The formation of disulphide bonds is a redox reaction. Proteins are oxidized by PDI when it is in an oxidized state, which produces a disulphide bond in the protein and renders PDI in a reduced state comprising free sulfhydryl groups.
ER oxidoreductin 1 protein (Ero1) is an essential component of oxidative folding machinery. Ero1 is a flavin adenine dinucleotide (FAD) dependent enzyme which facilitates disulphide bond formation in immunoglobulin subunits by selectively oxidizing PDI (Mezghrani et al., 2001, EMBO Journal, 20(22), 6288-6296). In humans two isoforms have been identified as human Ero1-Lα and Ero1-Lβ.
U.S. Pat. No. 6,361,964 discloses expression systems that make use of Ero1 to enhance disulphide bond formation and thereby to increase the yield of properly folded recombinant proteins.
The unfolded protein response (UPR) allows cells to respond to an increased demand on the protein folding capacity of the ER by coordinating the down regulation of protein synthesis with the increased expression of various proteins including ER resident chaperone proteins and folding enzymes which enable protein folding (Gunn et al., 2004, Molecular Immunology, 41, 919-9927). The UPR may also cause enlargement of the ER in order to provide increased capacity for protein folding.
X Box Binding Protein 1 (XBP1) is known to be a key regulator of the unfolded protein response. XBP1 is a transcription factor which is involved in B cell differentiation, ER expansion by stimulating activity of enzymes involved in lipid biosynthesis and regulates gene encoding ER resident chaperone proteins involved in the UPR (Lee et al., 2003, Molecular and Cellular Biology, 23(21), 7448-7459). XBP1 is believed to cause enlargement of the ER by up-regulating or activating enzymes including choline cytidylyltransferase (CCT). CCT is the rate-limiting enzyme in the CDP-choline pathway for the production of phosphatidylcholine (PtdCho), which is the primary phospholipid of the ER membrane.
The use of XBP1 in methods for protein production wherein a protein of interest is co-expressed with XBP1 are described in WO 2004/111194, WO 2006/028889 and US2005/0250182.
Accordingly, methods to improve the yield of protein expression systems using a component of the UPR pathway are known. However, there is still a need to provide improved methods for increasing the yield of proteins in expression systems.